Characterization of Particle Translocation through Mucin Hydrogels

Biological functional entities surround themselves with selective barriers that control the passage of certain classes of macromolecules while rejecting others. A prominent example of such a selective permeability barrier is given by mucus. Mucus is a biopolymer-based hydrogel that lines all wet epithelial surfaces of the human body. It regulates the uptake of nutrients from our gastrointestinal system, adjusts itself with the menstrual cycle to control the passage of sperm, and shields the underlying cells from pathogens such as bacteria and viruses. In the case of drug delivery, the mucus barrier needs to be overcome for successful medical treatment. Despite its importance for both physiology and medical applications, the underlying principles which regulate the permeability of mucus remain enigmatic. Here, we analyze the mobility of microscopic particles in reconstituted mucin hydrogels. We show that electrostatic interactions between diffusing particles and mucin polymers regulate the permeability properties of reconstituted mucin hydrogels. As a consequence, various parameters such as particle surface charge and mucin density, and buffer conditions such as pH and ionic strength, can modulate the microscopic barrier function of the mucin hydrogel. Our findings suggest that the permeability of a biopolymer-based hydrogel such as native mucus can be tuned to a wide range of settings in different compartments of our bodies.     

Diffusion of macromolecules and virus-like particles in human cervical mucus

To determine whether or not large macromolecules and viruses can diffuse through mucus, we observed the motion of proteins, microspheres, and viruses in fresh samples of human cervical mucus using fluorescent recovery after photobleaching and multiple image photography. Two capsid virus-like particles, human papilloma virus (55 nm, approximately 20,000 kDa) and Norwalk virus (38 nm, approximately 10,000 kDa), as well as most of the globular proteins tested (15-650 kDa) diffused as rapidly in mucus as in saline. Electron microscopy of cervical mucus confirmed that the mesh spacing between mucin fibers is large enough (20-200 nm) for small viruses to diffuse essentially unhindered through mucus. In contrast, herpes simplex virus (180 nm) colocalized with strands of thick mucus, suggesting that herpes simplex virus, unlike the capsid virus particles, makes low-affinity bonds with mucins. Polystyrene microspheres (59-1000 nm) bound more tightly to mucins, bundling them into thick cables. Although immunoglobulins are too small to be slowed by the mesh spacing between mucins, diffusion by IgM was slowed by mucus. Diffusion by IgM-Fc(5 mu), the Fc pentamer core of an IgM with all 10 Fab moieties removed, was comparably slowed by mucus. This suggests that the Fc moieties of antibodies make low-affinity bonds with mucins.     

Serine Protease(s) Secreted by the Nematode Trichuris muris Degrade the Mucus Barrier

The polymeric mucin component of the intestinal mucus barrier changes during nematode infection to provide not only physical protection but also to directly affect pathogenic nematodes and aid expulsion. Despite this, the direct interaction of the nematodes with the mucins and the mucus barrier has not previously been addressed. We used the well-established Trichuris muris nematode model to investigate the effect on mucins of the complex mixture of immunogenic proteins secreted by the nematode called excretory/secretory products (ESPs). Different regimes of T. muris infection were used to simulate chronic (low dose) or acute (high dose) infection. Mucus/mucins isolated from mice and from the human intestinal cell line, LS174T, were treated with ESPs. We demonstrate that serine protease(s) secreted by the nematode have the ability to change the properties of the mucus barrier, making it more porous by degrading the mucin component of the mucus gel. Specifically, the serine protease(s) acted on the N-terminal polymerising domain of the major intestinal mucin Muc2, resulting in depolymerisation of Muc2 polymers. Importantly, the respiratory/gastric mucin Muc5ac, which is induced in the intestine and is critical for worm expulsion, was protected from the depolymerising effect exerted by ESPs. Furthermore, serine protease inhibitors (Serpins) which may protect the mucins, in particular Muc2, from depolymerisation, were highly expressed in mice resistant to chronic infection. Thus, we demonstrate that nematodes secrete serine protease(s) to degrade mucins within the mucus barrier, which may modify the niche of the parasite to prevent clearance from the host or facilitate efficient mating and egg laying from the posterior end of the parasite that is in intimate contact with the mucus barrier. However, during a T_H2-mediated worm expulsion response, serpins, Muc5ac and increased levels of Muc2 protect the barrier from degradation by the nematode secreted protease(s).     

Spatial Configuration and Composition of Charge Modulates Transport into a Mucin Hydrogel Barrier

The mucus barrier is selectively permeable to a wide variety of molecules, proteins, and cells, and establishes gradients of these particulates to influence the uptake of nutrients, the defense against pathogens, and the delivery of drugs. Despite its importance for health and disease, the criteria that govern transport through the mucus barrier are largely unknown. Studies with uniformly functionalized nanoparticles have provided critical information about the relevance of particle size and net charge for mucus transport. However, these particles lack the detailed spatial arrangements of charge found in natural mucus-interacting substrates, such as certain viruses, which may have important consequences for transport through the mucus barrier. Using a novel, to our knowledge, microfluidic design that enables us to measure real-time transport gradients inside a hydrogel of mucins, the gel-forming glycoprotein component of mucus, we show that two peptides with the same net charge, but different charge arrangements, exhibit fundamentally different transport behaviors. Specifically, we show that certain configurations of positive and negative charges result in enhanced uptake into a mucin barrier, a remarkable effect that is not observed with either charge alone. Moreover, we show that the ionic strength within the mucin barrier strongly influences transport specificity, and that this effect depends on the detailed spatial arrangement of charge. These findings suggest that spatial charge distribution is a critical parameter to modulate transport through mucin-based barriers, and have concrete implications for the prediction of mucosal passage, and the design of drug delivery vehicles with tunable transport properties.     

Action of reactive oxygen species on colonic mucus secretions

Reactive oxygen species (ROS) have been implicated in the pathogenesis of many colonic diseases. Mucus is the colon's first line of defence against luminal agents. This study has therefore characterised ROS action on colonic mucus secretions. ROS were produced using peroxide-based systems of different concentrations. The effects of these systems were tested on native colonic mucus gels, isolated colonic mucins, and in vivo models. Colonic mucus gels were resistant to ROS breakdown. Mucins were susceptible to ROS attack, causing loss of terminal sugars and protein and mucin fragmentation. The in vivo thickness of the mucus barrier was reduced by up to 50% by ROS (above 5 mM peroxide). A 5 mM peroxide caused a significant increase in resting mucus thickness of ca. 15%. All ROS-generating systems caused mucosal damage once the loosely adherent mucus had been removed. As native mucus gel is more resistant to ROS damage than purified mucin, nonmucin components of mucus may have extensive ROS-scavenging properties. Low levels of luminal colonic ROS increase the protection afforded by the mucus barrier in vivo. Higher levels of ROS significantly reduce this protection. In vitro modeling of mucus degradation by ROS does not necessarily correlate with the dynamic, in vivo situation.     

Synthesis and macromolecular organization of gastrointestinal mucin.

Mucus glycoproteins (mucins), the principal determinants of mucus protective qualities and mucosal defense, are studied extensively to define pathological aberrations in the relation to gastrointestinal disease and to develop the mucous barrier strengthening agents. Recent work from our laboratory provided evidence as to the initial stages of the gastrointestinal mucin synthesis, molecular size of the apomucin, its macromolecular organization and interaction with other elements of gastrointestinal mucus. Using monoclonal antibodies against apomucin (clone 1H7), O-glycosylated with N-acetylgalactosamine apomucin (clone 2B4), and that against carboxyl terminal of the apomucin (clone 3G12), the mucin synthesizing polysomes were isolated and glycosylated peptides ranging in size from 6-60 kDa identified. The in vitro synthesis in the cell-free system also afforded 60-64 kDa products recognized by 1H7 and 3G12 antimucin MAbs. The obtained results provided evidence that the mucin core consists of 60 kDa peptide which at cotranslational stage is O-glycosylated with N-acetylgalactosamine. Studies on mucin polymer assembly revealed that mucin preparations prepared by equilibrium density gradient centrifugation and Sepharose 2B chromatography (Mantle, M., Mantle, D., and Allen, A. (1981) Biochem. J. 195, 277-285) are not completely purified and contain DNA and extraneous proteins. The evidence was obtained that so called mucin "link protein", 118 kDa glycopeptide, is a N-glycosylated fragment of fibronectin, whereas the supposedly native undegraded mucin isolated by Carlstedt et al. (Biochem. J. (1983) 211, 13-22) was found to contain mucin-fibronectin-DNA complexes. The general picture that emerged from the studies is that the pure mucin consists of 60 kDa glycosylated peptides only. The carboxyl terminal (8-12 kDa fragment) of these peptides is not glycosylated (naked) and is responsible for mucin interaction with fibronectin and other fibronectin-like extracellular matrix proteins. While the formation of the mucosal coat depends on many other factors and extracellular components, our findings on mucin structure and interaction with the extracellular matrix proteins provide explanation as to the possible mechanism of mucin adherence to the epithelial surfaces.     

Altering Mucus Rheology to “Solidify” Human Mucus at the Nanoscale

The ability of mucus to function as a protective barrier at mucosal surfaces rests on its viscous and elastic properties, which are not well understood at length scales relevant to pathogens and ultrafine environmental particles. Here we report that fresh, undiluted human cervicovaginal mucus (CVM) transitions from an impermeable elastic barrier to non-adhesive objects sized 1 µm and larger to a highly permeable viscoelastic liquid to non-adhesive objects smaller than 500 nm in diameter. Addition of a nonionic detergent, present in vaginal gels, lubricants and condoms, caused CVM to behave as an impermeable elastic barrier to 200 and 500 nm particles, suggesting that the dissociation of hydrophobically-bundled mucin fibers created a finer elastic mucin mesh. Surprisingly, the macroscopic viscoelasticity, which is critical to proper mucus function, was unchanged. These findings provide important insight into the nanoscale structural and barrier properties of mucus, and how the penetration of foreign particles across mucus might be inhibited.     

The rheology of pig small intestinal and colonic mucus: weakening of gel structure by non-mucin components.

Mechanical spectroscopy has been used to study the structure and properties of pig small intestinal and colonic adherent mucus gel. Both mucus secretions had properties of viscoelastic gels, but that from the small intestine was substantially weaker in quality. Small intestinal mucus gel was disrupted by acid (pH 1), detergents (bile) and protein denaturants while that from the colon remained stable following these treatments. Concentration of purified colonic mucin produced a gel with the same rheological properties as the native secretion. Purified small intestinal mucin when concentrated produced a stronger gel than the native secretion and, in contrast to the latter, one which was not disrupted by acid or denaturants. The instability of native small intestinal mucus was shown not to be a function of the mucin components (which alone could account for the gel-forming properties), but to arise from the presence of insoluble material largely from sloughed mucosal cells. These studies show (1) that mucus gels from the colon and small intestine have similar mechanical behaviour and properties to those from the stomach and duodenum, and (2) emphasise the caution that should be exercised when interpreting the rheological properties of mucus preparations, particularly with respect to their content of mucosal cellular material.     

Breakdown of mucin as barrier to digestive enzymes in the ischemic rat small intestine

Loss of integrity of the epithelial/mucosal barrier in the small intestine has been associated with different pathologies that originate and/or develop in the gastrointestinal tract. We showed recently that mucin, the main protein in the mucus layer, is disrupted during early periods of intestinal ischemia. This event is accompanied by entry of pancreatic digestive enzymes into the intestinal wall. We hypothesize that the mucin-containing mucus layer is the main barrier preventing digestive enzymes from contacting the epithelium. Mucin breakdown may render the epithelium accessible to pancreatic enzymes, causing its disruption and increased permeability. The objective of this study was to investigate the role of mucin as a protection for epithelial integrity and function. A rat model of 30 min splanchnic arterial occlusion (SAO) was used to study the degradation of two mucin isoforms (mucin 2 and 13) and two epithelial membrane proteins (E-cadherin and toll-like receptor 4, TLR4). In addition, the role of digestive enzymes in mucin breakdown was assessed in this model by luminal inhibition with acarbose, tranexamic acid, or nafamostat mesilate. Furthermore, the protective effect of the mucin layer against trypsin-mediated disruption of the intestinal epithelium was studied in vitro. Rats after SAO showed degradation of mucin 2 and fragmentation of mucin 13, which was not prevented by protease inhibition. Mucin breakdown was accompanied by increased intestinal permeability to FITC-dextran as well as degradation of E-cadherin and TLR4. Addition of mucin to intestinal epithelial cells in vitro protected against trypsin-mediated degradation of E-cadherin and TLR4 and reduced permeability of FITC-dextran across the monolayer. These results indicate that mucin plays an important role in the preservation of the mucosal barrier and that ischemia but not digestive enzymes disturbs mucin integrity, while digestive enzymes actively mediate epithelial cell disruption.     

<Emphasis Type="Italic">Listeria </Emphasis><Emphasis Type="Italic">monocytogenes</Emphasis> internalins bind to the human intestinal mucin MUC2

Listeria monocytogenes cross the intestinal barrier causing systemic infections with high mortality rates. Intestinal infection triggers release of intestinal mucus. We show that three L. monocytogenes internalins, InlB, InlC and InlJ all bound to MUC2 (the major component of intestinal mucus), but not to the cell surface mucin MUC1. Binding was strongest to InlB>InlC>InlJ (P < 0.001). Listerial internalins are characterized by their internalin domain, composed by leucine rich repeats (LRR) followed by an immunogloblin-like region. We report here that the internalin domain of the InlJ protein also bound MUC2, suggesting that an internalin domain is sufficient to bind to MUC2.     

Mucin gene expression in rat airways following infection and irritation.

Airway mucus hypersecretion occurs in response to infection and irritation and poses an important and poorly understood clinical problem. In order to gain insight into its pathogenesis, we have focused on an mRNA encoding the major mucus glycoprotein, mucin. Northern blots showed that mucin mRNA was abundant in the intestine of specific pathogen free rats whereas it was undetectable in the airways of these rats until pathogen-free conditions were suspended and rats acquired Sendai (Parainfluenza I) virus infections. Airway mucin hybridization signals in rats that were both infected with Sendai virus and exposed to SO2 were more intense than those in rats with infection alone. These results suggest that pathogen-and irritant-induced hypersecretion may be partly controlled at the level of mucin mRNA.     

Quantitative MUC5AC and MUC6 mucin estimations in gastric mucus by a least-squares minimization method

We have determined the molar proportions of the MUC5AC and MUC6 mucus glycoproteins (mucins) in mucus from the normal and pathological human gastric antrum using a least-squares minimization analysis applied to amino acid compositions. We noted that the content of MUC5AC mucin in mucus from individuals without gastroduodenal disease was very high, suggesting that the integrity and barrier properties of the adherent gastric mucus layer are normally maintained by building-block structures formed from this mucin alone. We observed that the molar content of MUC6 mucin doubled (without significance) in mucus from patients with duodenal ulcer, and increased five times (with high significance) in mucus from patients with gastric ulcer, when compared with that in mucus from individuals without gastroduodenal disease.     

Apoptosis: a mechanism of cell killing by influenza A and B viruses.

In previous studies, we observed that the virulent avian influenza A virus A/Turkey/Ontario/7732/66 (Ty/Ont) induced severe lymphoid depletion in vivo and rapidly killed an avian lymphocyte cell line (RP9) in vitro. In examining the mechanism of cell killing by this virus, we found that Ty/Ont induced fragmentation of the RP9 cellular DNA into a 200-bp ladder and caused ultrastructural changes characteristic of apoptotic cell death by 5 h after infection. We next determined that the ability to induce apoptosis was not unique to Ty/Ont. In fact, a variety of influenza A viruses (avian, equine, swine, and human), as well as human influenza B viruses, induced DNA fragmentation in a permissive mammalian cell line, Madin-Darby canine kidney (MDCK), and this correlated with the development of a cytopathic effect during viral infection. Since the proto-oncogene bcl-2 is a known inhibitor of apoptosis, we transfected MDCK cells with the human bcl-2 gene; these stably transfected cells (MDCKbcl-2) did not undergo DNA fragmentation after virus infection. In addition, cytotoxicity assays at 48 to 72 h after virus infection showed a high level of cell viability for MDCKbcl-2 compared with a markedly lower level of viability for MDCK cells. These studies indicate that influenza A and B viruses induce apoptosis in cell cultures; thus, apoptosis may represent a general mechanism of cell death in hosts infected with influenza viruses.     

Somatostatin stimulates colonic MUC2 expression through SSTR5-Notch-Hes1 signaling pathway

Colonic mucus barrier is regarded as the first defense line against bacteria and antigens from directly attaching to the epithelium, which would further lead to intestinal inflammation activation and pathological conditions. As MUC2 mucin is the predominant component of the mucus, understanding the regulatory mechanisms of MUC2 is important for mucus barrier protection. Somatostatin (SST) has been found to play a role in colon protection through various manners. However, whether SST involves in colonic mucus barrier regulation is still unclear. The aim of this study is to investigate the effects and potential mechanisms of SST on colonic MUC2 expression and mucus secretion. In vivo study, exogenous somatostatin (octreotide) administration effectively stimulated mice colonic MUC2 expression and mucus secretion. In human goblet-like cell LS174T cells, SST exposure also significantly stimulated MUC2 expression and mucus secretion. Further studies indicated that SST receptor 5 (SSTR5) was significantly activated by SST, whereas specific SSTR5 siRNA transfection of LS174T cells significantly blocked SST-induced increase in MUC2 expression and mucus secretion. In addition, SSTR5 agonist L817,818 also upregulated MUC2 expression and mucus secretion in LS174T cells. Mechanistic studies further demonstrated that SST/SSTR5-mediated MUC2 upregulation was dependent on Notch-Hes1 pathway suppression by detecting notch intracellular domain (NICD) and Hes1 proteins. Taken together, our findings suggested that SST could participate in colonic mucus barrier regulation through SSTR5-Notch-Hes1-MUC2 signaling pathway. These findings provide a deep insight into the role of SST on colonic mucus regulation under physiological conditions.     

Sensitization of rhabdo-, lenti-, and spumaviruses to human serum by galactosyl(alpha1-3)galactosylation.

Vesicular stomatitis virus, human immunodeficiency virus type 2, and human foamy virus, which were produced by cell lines expressing galactosyl(alpha1-3)galactosyl (alphaGal) sugars, were found to be less stable in human serum than those from alphaGal-negative cells, indicating that galactosyl(alpha1-3)galactosylation sensitizes these viruses as well as mammalian type C oncoviruses (Rother et al., J. Exp. Med. 182:1345-1355, 1995; Takeuchi et al., Nature (London) 379:85-88, 1996) to complement killing via natural anti-alphaGal antibodies. Thus, virus killing mediated by anti-alphaGal antibodies may play a role as a barrier to animal-to-human infection of various enveloped viruses. Virus vectors for human in vivo gene therapy based on the viruses mentioned above should be produced from alphaGal-negative cells.     

Identification of human immunodeficiency virus envelope gene sequences influencing viral entry into CD4-positive HeLa cells, T-leukemia cells, and macrophages.

Infectious recombinant viruses were constructed from three molecularly cloned human immunodeficiency virus (HIV) strains varying in cell tropism. All recombinants showed a high infectivity titer on phytohemagglutinin-stimulated normal T lymphocytes. However, a 120-bp region of the envelope gene including the area of the V3 hypervariable loop was found to influence infectivity titer on both clone 1022 CD4-positive HeLa cells and CD4-positive CEM leukemia cells. Infectivity for macrophages was more complex. All viruses replicated in macrophages to a low level, but viral sequences both inside and outside the V3 loop region influenced the efficiency of replication. Two experiments showed that the mechanism of restriction of infection of 1022 cells by HIV strain JR-CSF was related to lack of virus entry. First, productive virus infection occurred after transfection of 1022 cells with viral plasmid DNA. Second, the nonpermissive HIV strain JR-CSF could infect 1022 cells when pseudotyped with the envelope of other retroviruses, including human T-cell leukemia virus type I (HTLV-I), HTLV-II, and amphotropic murine leukemia virus. These results demonstrate the possibility that unexpected cell types might be infected with HIV in human patients coinfected with HIV and HTLV-I or HTLV-II.     

Avian influenza virus

Recent outbreaks of highly pathogenic avian influenza in chickens and ducks that occurred in 9 Asian countries including Japan alarmed to realize that there is no border for infections and gave a rise to great concern for human health as well as for agriculture. This H5N1 virus jumped the species barrier and caused severe disease with high mortality in humans in Viet Nam and Thailand; 15 deaths of 22 cases and 8 of 12, respectively. A second concern was the possibility that the situation could give rise to another influenza pandemic in humans since genetic reassortment may occur between avian and human influenza viruses when a person is concurrently infected with viruses from both species. This process of gene swapping inside the human body can give rise to a new subtype of the influenza virus to which humans would not have immunity. The outbreaks also emphasized the need to continue active surveillance on avian influenza throughout the year to undertake aggressive emergency control measures as soon as an infection is detected.     

Effect of MMP/ADAM inhibitors on goblet cell hyperplasia in cultured human bronchial epithelial cells

While epidermal growth factor receptor (EGFR) plays a pivotal role in the repair process of epithelial cells, it is also involved in the overproduction of mucus and goblet cell hyperplasia (GCH), which occurs in chronic airway diseases such as asthma. Among the EGFR ligands, transforming growth factor (TGF)-alpha is thought to be the most important in the synthesis of mucus. Pro-TGF-alpha is cleaved to give an active form by members of the matrix metalloproteinases (MMP)/a disintegrin and metalloproteinases (ADAM) family. Thus MMP/ADAM inhibitors might prevent GCH by inhibiting transactivation of EGFR. Upon stimulation of differentiating normal human bronchial epithelial (NHBE) cells by IL-13, GCH was induced. The mucin genes MUC5AC, MUC5B, and MUC2 were upregulated whereas the expression of ciliated cell markers was greatly repressed. GM6001, a broad-spectrum inhibitor for MMP/ADAM, inhibited IL-13-induced mucin gene expression and mucus production as measured by periodic acid-Schiff staining. This was accompanied by an inhibition of TGF-alpha release. These results suggest that MMP/ADAMs play a pivotal role in the development of GCH in lung epithelial cells.